The overall objective of this proposal is an understanding of the role and affects of the short, interspersed repeats (SINEs) in the mammalian genome. This also includes a better understanding of the RNA mediated transposition mechanism (retroposition) which spreads them throughout the genome. We will continue to carry out characterizations of the RNA polymerase III promoter of the SINEs and factors that affect its activity. These studies will primarily be carried out on recombinant DNA constructs introduced back into culture cells. We will study the affect of nearby sequences, such as enhancers and RNA polymerase II promoters, on the SINE promoter, and more general factors such as chromatin structure and methylation. A second line of experimentation will involve determination of the affect that the SINEs have on increasing both homologous and illegitimate recombinations in the mammalian genome. These studies will involve introduction of recombinant constructs into cells and assaying for recombination using selection techniques. This will be carried out by making human thymidine kinase minigenes which are defective unless the recombination has occurred. We will sequence specific regions (the thymidine kinase region) in several primate genomes in order to determine the time course of appearance of the Alu family in primates. Analysis of these sequences will show when Alu spread through the genome and whether it is still spreading. It will also answer the question of whether there are mechanisms removing Alu family members, as well as inserting them. Additionally we will analyze site- specificity of the integration of the Alu family sequences. We will make recombinant constructs that allow us to prepare RNA molecules that correspond to proposed intermediates in the retroposition process. We will use these intermediates to test some of the retroposition mechanisms parameters in vitro and eventually within transgenic mice.